Evaluation of Jaw Osteonecrosis Following Intra-Ligament Anesthesia in Zoledronate-Treated Rats: a Histological Evaluation

Statement of the Problem: One of the rare adverse effects in patients who take bisphosphonates is the osteonecrosis of the jaw in the oral cavity following any trauma such as tooth extraction. Purpose: The aim of this study is the histopathological evaluation of the jaw following intra-ligament anesthesia injection in Zoledronate-treated rats. Materials and Method: In this descriptive-experimental study, rats weighing 200-250 g were divided into 2 groups. The first group received a 0.6 mg/kg dose of zoledronate and the second group received normal saline. Five injections with a 28-day interval were performed. At the end of the injection, the animals were sacrificed. Then, five-micrometer histological slides were prepared from the first maxillary molars and the surrounding tissues. Hematoxylin and eosin staining was performed to evaluate osteonecrosis, infiltration of inflammatory cells, fibrosis, and root and bone resorption. Results: There was no difference between the macroscopic and clinical features in both groups and no evidence of osteonecrosis of the jaw was observed in the samples. From the histological point of view, all the samples had normal tissues and none of them showed any evidence of inflammation, tissue fibrosis, disorder, or pathological root resorption. Conclusion: According to the histological findings, the periodontal ligament space, the bone adjacent to the roots, and the dental pulp conditions were similar in both groups. Osteonecrosis of the jaw did not develop in the rats that took bisphosphonates after intraligamental injection.


Introduction
As a group of antiresorptive agents, bisphosphonates are used in bone diseases with high bone resorption such as bone malignancy (to prevent metastasis), osteoporosis, multiple myeloma, Paget's disease, and hypercalcemia [1]. These drugs induce apoptosis in osteoclasts and inhibit their activity. Moreover, they decrease bone resorption and increase bone mineralization [2]. Bisphosphonates have a high affinity for the skeleton and accu-mulate in active bone remodeling sites [3]. They remain in the bone for a long time and have detrimental effects on it. Zoledronate is a highly potent drug and belongs to the third generation of bisphosphonates [4]. The osteonecrosis of the jaw is one of the rare adverse effects of these drugs and can happen after traumatic procedures such as dental extraction and maxillofacial surgery [5].
One of the important advancements in dental therapeutics is the development of safe and effective local anes-thetic techniques. However, anesthetic effectiveness is sometimes poor especially after a mandibular or maxillary nerve block in patients with severe inflammation in the dental pulp [6]. For these patients, some supplemental anesthetic techniques are available. In intraligament anesthesia, a high injection pressure is used to force the local anesthetic solution through the periodontal ligament into the cancellous medullary bone surrounding the tooth [7].
Intra-ligament anesthesia may cause injury and trauma to the periodontal ligament and the surrounding tissues. Hence, the aim of this study was a histological evaluation of intra-ligament injection in zoledronate-treated rats to assess the osteonecrosis of the jaw in them.

Materials and Method
The protocol of this study was reviewed and approved One hundred fifty days after the start of the study, the animals were subjected to vital perfusion and the first molar teeth and the surrounding tissues were separated from the maxilla. First, the samples were fixed in formalin 10% for 24 hours. Afterwards, calcification was performed with 85% formic acid for one week and the samples were embedded in paraffin. Subsequently, 5-µm-thick sections were prepared and used for hematoxylin and eosin staining. A histological examination was performed to evaluate osteonecrosis, infiltration of inflammatory cells, fibrosis, and root and bone resorption.

Results
The histological evaluation was performed for the two groups (zoledronate and saline therapies). There was no evidence of wound and defect in the maxilla of the rats that received zoledronate and intra-ligament injection

Discussion
Bisphosphonates are non-metabolic drugs and pyrophosphate analogues, which prevent the transition of amorphous calcium to hydroxyapatite and the accumulation of calcium phosphate crystals [9]. Their most important clinical effect is the prevention of bone resorption. In their non-metabolized form, bisphosphonates remain in the bone over a long period of time. The activity of osteoclasts is reduced following the administration of these drugs [9]. One of the rare adverse effects of bisphosphonates is the osteonecrosis of the jaw, which can occur following injuries in the jaw such as tooth extraction [10]. Intraosseous injection may cause osteonecrosis of the jaw in patients who take bisphosphonates [10,13]. Incomplete anesthesia is one of the complications in the process of anesthesia and requires the use of supplemental anesthetic techniques. The intra-ligament anesthetic technique is a common supplemental anesthetic method [10][11][12][13]. Therefore, this study has been organized to investigate the relationship between bisphosphonate-related osteonecrosis of the jaw and intra-ligament injection. Although intra-ligament injection in the periodontal ligament space results in a suitable numbness, it has some adverse effects including decreased local blood flow, periodontal tissue destruction, and localized inflammation [11][12][13].
In the present study, no differences were observed in the histological samples of the treated and control groups. In addition, no sign of fibrosis was observed in the histological samples. On the other hand, in vitro studies have shown that zoledronate decreases the cell viability of both periodontal ligament fibroblasts [14][15][16] and tumor cells [17][18]. Allam et al. [19] reported that zoledronate had toxic effects on dental pulp cells in micro-molar concentrations, reducing their proliferation.
The onset of the bisphosphonate-related osteonecrosis of the jaw may be due to dental pulp necrosis.
This study revealed no differences between the two groups. The significant difference between the in vitro and in vivo results is due to the different conditions. Bisphosphonates reduce bone remodeling; prevent osteoclast activity, decrease bone loss, and increase bone density [20][21].
Pourgonabadi et al. [22] showed that zoledronate has anti-proliferative and pro-apoptotic effects in the dental pulp stem cells (DPSCs), which may lead to zole- In the current study, no accumulation of zoledronate was observed in the dental tissues. A proportion of zoledronate is released during the bone remodeling process and the drug is likely to be exposed to the environment [25]. Basso et al. [26] showed that zoledronate had a significant effect on the reduction of the number of viable cells as well as the viability of epithelial cells (by 70%) and fibroblasts (by 40%). This revealed the toxicity of zoledronate for these cells. Agis et al. [14] evaluated the effects of zoledronate with a concentration of 30 μM in fibroblast cultures. Their results demonstrated the reduction of cell viability and proliferation in the fibroblasts. The differences in the results of various studies can be due to the different concentrations of zoledronate used in them. This is consistent with some studies that have reported the dose-dependent toxicity of bisphosphonate medications [15].
In this study, the evaluation of the cellular morphology of the dental and periodontal tissues did not show any significant differences between the treated and control groups. The results of the histological evaluation of the dental pulp and the surrounding tissues showed no significant differences between the control and treated groups. According to Basso et al. [26], the incidence of cell death was evident for some epithelial cells and fibroblasts with the separation of the glass substrate in the zoledronate-treated patients, although most of the cells adhered to the substrate and showed significant morphological changes. They also indicated that zoledronate (with a concentration of 5 μM) was toxic for epithelial cells and fibroblasts [26]. These results cannot be generalized to clinical conditions. In addition, the effects of bisphosphonates in the oral cavity remain unknown. In vitro and in vivo studies have demonstrated that bisphosphonates prevent the function of endothelial cells [27].
By reducing the blood flow, bisphosphonates play an antiangiogenic role in the necrosis areas [28]. Another cause of the osteonecrosis of the jaw is the function of surgical processes [28]. Osteoclasts have adverse effects on the wound healing process by reducing cytokines in the bone marrow and bone surfaces, increasing the risk of osteomyelitis and necrosis [29]. Both jaw bones have a high rate of remodeling due to their mechanical ability in mastication. It is assumed that bisphosphonates accumulate in the bone and are simultaneously toxic for the oral cavity. They prevent soft tissue healing by causing secondary infections under bone tissues [29].

Conclusion
According to the histological findings, the periodontal ligament space, the bone adjacent to the roots, and the dental pulp conditions were similar in both groups. Osteonecrosis of the jaw did not develop in the rats that took bisphosphonates after intraligamental injection.